The receptor tyrosine kinase EphB4 is a highly attractive angiogenic target involved in many types of cancer (Cheng N., Brantley D. M. and Chen J., Cytokine Growth Factor Rev 2002, 13:75-85). Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a multi-step process involving many various factors, which stimulate endothelial cell proliferation, migration, and assembly, as well as recruitment of perivascular cells and extracellular matrix remodelling. Angiogenesis is implicated in the pathogenesis of a variety of disorders, including solid tumours, intraocular neovascular syndromes such as proliferative retinopathies or age-related macular degeneration (AMD), rheumatoid arthritis, and psoriasis.
Ephrins and their receptors were first identified in studies for neuronal growth during development. Unlike other families of receptor tyrosine kinases, which bind soluble ligands, ephrin receptors interact with cell surface-bound ephrin ligands. Ephrins attach to the cell membrane either through a glycosylphosphatidyl inositol anchor (ephrin A) or a transmembrane domain (ephrin B). Ephrin receptors are likewise divided in two subclasses EphA and EphB, depending on the type of interaction with their ligands ephrin A or B. Ephrins and their receptors have been shown to play an essential role in vascular development during embryogenesis and in adult angiogenesis, as key regulators of vascular assembly, arteriovenous differentiation, and boundary formation. Both EphA and EphB receptors and their ligands are involved in vascular development. Especially, ephrin B2 is expressed in arterial endothelial cells, whereas its cognate receptor, EphB4 is expressed in venous endothelial cells. EphB4 (also named HTK) and its ligand, ephrin B2 (HTKL), play important roles in establishing and determining vascular networks. Blocking this receptor/ligand pair inhibits the end stage of tumour blood vessel formation, as well as causes direct growth inhibition of certain cancers.
EphB4 seems to be rather recalcitrant to inhibition because, despite its potential therapeutic importance, only two series of non-peptidic small molecule inhibitors have been reported in the literature up to date (Miyazaki Y. et al., Bioorg Med Chem Lett 2007, 17:250-254). Examples of angiogenesis inhibitors are found in WO 2006/131003 and WO 2007/062805.
Docking is an important and successful method in computer-aided drug design. However, there is a large and increasing discrepancy between the number of small molecules available in computer-readable format (in the order of 107-108 compounds, not counting those generated by de novo design programs) and the amount of molecules that can be efficiently processed by high-throughput approaches in silico or in vitro (in the order of 105-106). Moreover, it is highly inefficient to use computational resources or robotic systems and chemical reagents for compounds that have a low chance of binding to the target protein. The essential question is how to utilize (structural) information of the target to pre-select the molecules that are most likely to show binding and inhibitory activity.
Several in silico fragment-based approaches, e.g., SEED, MCSS and FFLD, and in vitro “needle screening” procedures have been suggested in the prior art. Needle- or anchor-based in vitro screening has been applied to a wide range of enzymes including thrombin, DNA gyrase, and protein tyrosine kinases. In addition, others have tried to reduce docking computation times by selecting ligands through the application of pharmacophore constraints and were able to identify inhibitors of enzymes.